After discovery of RSV in the 1950s, the virus soon became a recognized pathogen associated with lower and upper respiratory tract infections in humans. Worldwide, it is estimated that 64 million RSV infections occur each year resulting in 160.000 deaths (WHO Acute Respiratory Infections Update September 2009). The most severe disease occurs particularly in premature infants, the elderly and immunocompromised individuals. In children younger than 2 years, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of the hospitalizations due to respiratory infections, and the peak of hospitalization occurs at 2-4 months of age. It has been reported that almost all children have been infected by RSV by the age of two. Repeated infection during lifetime is attributed to ineffective natural immunity. The level of RSV disease burden, mortality and morbidity in the elderly are second to those caused by nonpandemic influenza A infections.
RSV is a paramyxovirus, belonging to the subfamily of pneumovirinae. Its genome encodes for various proteins, including the membrane proteins known as RSV Glycoprotein (G) and RSV fusion (F) protein which are the major antigenic targets for neutralizing antibodies. Proteolytic cleavage of the fusion protein precursor (F0) yields two polypeptides F1 and F2 linked via disulfide bridge. Antibodies against the fusion-mediating part of the F1 protein can prevent virus uptake in the cell and thus have a neutralizing effect. Besides being a target for neutralizing antibodies, RSV F contains cytotoxic T cell epitopes (Pemberton et al., 1987, J. Gen. Virol. 68: 2177-2182).
Treatment options for RSV infection include a monoclonal antibody against the F protein of RSV. The high costs associated with such monoclonal antibodies and the requirement for administration in a hospital setting, preclude their use for prophylaxis in the at-risk population at large scale. Thus, there is a need for an RSV vaccine, which preferably can be used for the pediatric population as well as for the elderly.
Despite 50 years of research, there is still no licensed vaccine against RSV. One major obstacle to the vaccine development is the legacy of vaccine-enhanced disease in a clinical trial in the 1960s with a formalin-inactivated (FI) RSV vaccine. FI-RSV vaccinated children were not protected against natural infection and infected children experienced more severe illness than non-vaccinated children, including two deaths. This phenomenon is referred to as “enhanced disease.”
Since the trial with the FI-RSV vaccine, various approaches to generate an RSV vaccine have been pursued. Attempts include classical live attenuated cold passaged or temperature sensitive mutant strains of RSV, (chimeric) protein subunit vaccines, peptide vaccines and RSV proteins expressed from recombinant viral vectors. Although some of these vaccines showed promising pre-clinical data, no vaccine has been licensed for human use due to safety concerns or lack of efficacy.
Adenovirus vectors are used for the preparation of vaccines for a variety of diseases, including disease associated with RSV infections. The following paragraphs provide examples of adenovirus-based RSV candidate vaccines that have been described.
In one approach, RSV.F has been inserted into the non-essential E3 region of replication competent adenovirus types 4, 5, and 7. Immunization in cotton rats, intranasal (i.n.) application of 107 pfu, was moderately immunogenic, and protective against lower respiratory tracts against RSV challenge, but not protective against upper respiratory tract RSV challenge (Connors et al., 1992, Vaccine 10: 475-484; Collins, P. L., Prince, G. A., Camargo, E., Purcell, R. H., Chanock, R. M. and Murphy, B. R. Evaluation of the protective efficacy of recombinant vaccinia viruses and adenoviruses that express respiratory syncytial virus glycoproteins. In: Vaccines 90: Modern Approaches to New Vaccines including prevention of AIDS (Eds. Brown, F., Chanock, R. M., Ginsberg, H. and Lerner, R. A.) Cold Spring Harbor Laboratory, New York, 1990, pp 79-84). Subsequent oral immunization of a chimpanzee was poorly immunogenic (Hsu et al., 1992, J Infect Dis. 66:769-775).
In other studies (Shao et al., 2009, Vaccine 27: 5460-71; U.S. 2011/0014220), two recombinant replication incompetent adenovirus 5 vectors carrying nucleic acid encoding the transmembrane truncated (rAd-F0ΔTM) or full-length (rAd-F0) version of the F protein of the RSV-B1 strain were engineered and given via the intranasal route to BALB/c mice. Animals were primed i.n. with 107 pfu and boosted 28 days later with the same dose i.n. Although the anti-RSV-B1 antibodies were neutralizing and cross-reacting with RSV-Long and RSV-A2 strain, immunization with these vectors protected only partially against RSV B1 challenge replication. The (partial) protection with rAd-F0ΔTM was slightly higher than with rAd-F0.
In another study, it was observed that BALB/c mice i.n. immunization with 1011 virus particles with the replication deficient (Ad5 based) FG-Ad adenovirus expressing wild type RSV F (FG-Ad-F) reduced lung viral titers only a 1.5 log 10 compared with the control group (Fu et al., 2009, Biochem. Biophys. Res. Commun. 381: 528-532.
In yet other studies, it was observed that intranasally applied recombinant Ad5-based replication-deficient adenovector expressing codon optimized soluble F1 fragment of F protein of RSV A2 (amino acid 155-524) (108 PFU) could reduce RSV challenge replication in the lungs of BALB/c mice compared to control mice, but mice immunized by the intramuscular (i.m.) route did not exhibit any protection from the challenge (Kim et al., 2010, Vaccine 28: 3801-3808).
In other studies, adenovectors Ad5-based carrying the codon optimized full-length RSV F (AdV-F) or the soluble form of the RSF F gene (AdV-Fsol) were used to immunize BALB/c mice twice with a dose of 1×1010 OPU (optical particle units: a dose of 1×1010 OPU corresponds with 2×108 GTU (gene transduction unit)). These vectors strongly reduced viral loads in the lungs after i.n. immunization, but only partially after subcutaneous (s.c.) or i.m. application (Kohlmann et al., 2009, J Virol 83: 12601-12610; U.S. 2010/0111989).
In yet other studies, it was observed that intramuscular applied recombinant Ad5-based replication-deficient adenovector expressing the sequenced F protein cDNA of RSV A2 strain (1010 particle units) could reduce RSV challenge replication only partially in the lungs of BALB/c mice compared to control mice (Krause et al., 2011, Virology Journal 8:375-386)
Apart from not being fully effective in many cases, the RSV vaccines under clinical evaluation for pediatric use and most of the vaccines under pre-clinical evaluation, are intranasal vaccines. The most important advantages of the intranasal strategy are the direct stimulation of local respiratory tract immunity and the lack of associated disease enhancement. Indeed, generally the efficacy of, for instance, the adenovirus based RSV candidate vaccines appears better for intranasal administration as compared to intramuscular administration. However, intranasal vaccination also gives rise to safety concerns in infants younger than 6 months. Most common adverse reactions of intranasal vaccines are runny nose or nasal congestion in all ages. Newborn infants are obligate nasal breathers and thus must breathe through the nose. Therefore, nasal congestion in an infant's first few months of life can interfere with nursing, and in rare cases can cause serious breathing problems.
More than 50 different human adenovirus serotypes have been identified. Of these, adenovirus serotype 5 (Ad5) has historically been studied most extensively for use as gene carrier. Recombinant adenoviral vectors of different serotypes may however give rise to different results with respect to induction of immune responses and protection. For instance, WO 2012/021730 describes that simian adenoviral vector serotype 7 and human adenoviral vector serotype 5 encoding F protein provide better protection against RSV than a human adenoviral vector of serotype 28. In addition, differential immunogenicity was observed for vectors based on human or non-human adenovirus serotypes (Abbink et al., 2007, J Virol 81: 4654-4663; Colloca et al., 2012, Sci Transl Med 4, 115ra2). Abbink et al., conclude that all rare serotype human rAd vectors studied were less potent than rAd5 vectors in the absence of anti-Ad5 immunity. Further it has been recently described that, while rAd5 with an Ebolavirus (EBOV) glycoprotein (gp) transgene protected 100% of non-human primates, rAd35 and rAd26 with EBOV gp transgene provided only partial protection and a heterologous prime-boost strategy was required with these vectors to obtain full protection against ebola virus challenge (Geisbert et al., 2011, J Virol 85: 4222-4233). Thus, it is a priori not possible to predict the efficacy of a recombinant adenoviral vaccine, based solely on data from another adenovirus serotype.
Moreover, for RSV vaccines, experiments in appropriate disease models such as cotton rat are required to determine if a vaccine candidate is efficacious enough to prevent replication of RSV in the nasal tract and lungs and at the same time is safe, i.e., does not lead to enhanced disease. Preferably such candidate vaccines should be highly efficacious in such models, even upon intramuscular administration.